Insights into the modes of action of tritium on the early-life stages of zebrafish, Danio rerio, using transcriptomic and proteomic analyses.

In the environment, populations are exposed to different kinds of ionizing radiation. Little is known about their modes of action on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, by exposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified, and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defence response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study, therefore, brought a body of evidence on the effects of HTO observed at the molecular level on zebrafish larvae. Further studies could investigate if the effects persist in adult organisms.

Tritium and Carbon-14 Contamination Reshaping the Microbial Community Structure, Metabolic Network, and Element Cycle in the Seawater Environment.

The potential ecological risks caused by entering radioactive wastewater containing tritium and carbon-14 into the sea require careful evaluation. This study simulated seawater's tritium and carbon-14 pollution and analyzed the effects on the seawater and sediment microenvironments. Tritium and carbon-14 pollution primarily altered nitrogen and phosphorus metabolism in the seawater environment. Analysis by 16S rRNA sequencing showed changes in the relative abundance of microorganisms involved in carbon, nitrogen, and phosphorus metabolism and organic matter degradation in response to tritium and carbon-14 exposure. Metabonomics and metagenomic analysis showed that tritium and carbon-14 exposure interfered with gene expression involving nucleotide and amino acid metabolites, in agreement with the results seen for microbial community structure. Tritium and carbon-14 exposure also modulated the abundance of functional genes involved in carbohydrate, phosphorus, sulfur, and nitrogen metabolic pathways in sediments. Tritium and carbon-14 pollution in seawater adversely affected microbial diversity, metabolic processes, and the abundance of nutrient-cycling genes. These results provide valuable information for further evaluating the risks of tritium and carbon-14 in marine environments.

Changes in the Structure of Potato Virus A Virions after Limited in situ Proteolysis According to Tritium Labeling Data and Computer Simulation.

Coat proteins (CP) of the potato virus A virions (PVA) contain partially disordered N-terminal domains, which are necessary for performing vital functions of the virus. Comparative analysis of the structures of coat proteins (CPs) in the intact PVA virions and in the virus particles lacking N-terminal 32 amino acids (PVAΔ32) was carried out in this work based on the tritium planigraphy data. Using atomic-resolution structure of the potato virus Y potyvirus (PVY) protein, which is a homolog of the CP PVA, the available CP surfaces in the PVY virion were calculated and the areas of intersubunit/interhelix contacts were determined. For this purpose, the approach of Lee and Richards [Lee, B., and Richards, F. M. (1971) J. Mol. Biol., 55, 379-400] was used. Comparison of incorporation profiles of the tritium label in the intact and trypsin-degraded PVAΔ32 revealed position of the ΔN-peptide shielding the surface domain (a.a. 66-73, 141-146) and the interhelix zone (a.a. 161-175) of the PVA CP. Presence of the channels/cavities was found in the virion, which turned out to be partially permeable to tritium atoms. Upon removal of the ΔN-peptide, decrease in the label incorporation within the virion (a.a. 184-200) was also observed, indicating possible structural transition leading to the virion compactization. Based on the obtained data, we can conclude that part of the surface ΔN-peptide is inserted between the coils of the virion helix thus increasing the helix pitch and providing greater flexibility of the virion, which is important for intercellular transport of the viruses in the plants.

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation.

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

Histamine H2 receptor radioligands: triumphs and challenges.

Since the discovery of the histamine H2 receptor (H2R), radioligands were among the most powerful tools to investigate its role and function. Initially, radiolabeling was used to investigate human and rodent tissues regarding their receptor expression. Later, radioligands gained increasing significance as pharmacological tools in in vitro assays. Although tritium-labeling was mainly used for this purpose, labeling with carbon-14 is preferred for metabolic studies of drug candidates. After the more-or-less successful application of numerous labeled H2R antagonists, the recent development of the G protein-biased radioligand [3H]UR-KAT479 represents another step forward to elucidate the widely unknown role of the H2R in the central nervous system through future studies.

Functional Expression of Choline Transporters in Human Neural Stem Cells and Its Link to Cell Proliferation, Cell Viability, and Neurite Outgrowth.

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

Protection of dopamine neurons by CDNF and neurturin variant N4 against MPP+ in dissociated cultures from rat mesencephalon.

Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 µM) decreased tyrosine hydroxylase-positive cells to 41.7 ± 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 ± 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 ± 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.

Transport and Toxicity of Methylmercury-Cysteine in Cultured BeWo Cells.

Mercury is a heavy metal toxicant that is prevalent throughout the environment. Organic forms of mercury, such as methylmercury (MeHg), can cross the placenta and can lead to lasting detrimental effects in the fetus. The toxicological effects of MeHg on the placenta itself have not been clearly defined. Therefore, the purpose of the current study was to assess the transport of MeHg into placental syncytiotrophoblasts and to characterize the mechanisms by which MeHg exerts its toxic effects. Cultured placental syncytiotrophoblasts (BeWo) were used for these studies. The transport of radioactive MeHg was measured to identify potential mechanisms involved in the uptake of this compound. The toxicological effects of MeHg on BeWo cells were determined by assessing visible pathological change, autophagy, mitochondrial viability, and oxidative stress. The findings of this study suggest that MeHg compounds are transported into BeWo cells primarily by sodium-independent amino acid carriers and organic anion transporters. The MeHg altered mitochondrial function and viability, decreased mitophagy and autophagy, and increased oxidative stress. Exposure to higher concentrations of MeHg inhibited the ability of cells to protect against MeHg-induced injury. The findings show that MeHg is directly toxic to syncytiotrophoblasts and may lead to disruptions in the fetal/maternal transfer of nutrients and wastes.

Region-specific and dose-specific effects of chronic haloperidol exposure on [3H]-flumazenil and [3H]-Ro15-4513 GABAA receptor binding sites in the rat brain.

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABAAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.

Cysteine redox state regulates human ß2-adrenergic receptor binding and function.

Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. ß2-adrenergic receptor (ß2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that ß2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes ß2AR cysteine thiols to Cys-S-sulfenic acids (Cys-S-OH). Furthermore, highly oxidative environments can irreversibly oxidize Cys-S-OH to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participating in homeostatic redox reactions (i.e., redox-deficient). The aim of this study was to examine the vitality of ß2AR-ROS interplay and the resultant functional consequences of ß2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that ß2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing ß2AR, we show that receptor redox state profoundly influences ß2AR orthosteric ligand binding and downstream function. Specifically, homeostatic ß2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to ß-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient ß2AR states exhibit decreased ability to signal via either Gαs or ß-arrestin. Together, our results demonstrate a ß2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.

Transport characteristics of 5-aminosalicylic acid into colonic epithelium: Involvement of sodium-coupled monocarboxylate transporter SMCT1-mediated transport system.

5-Aminosalicylic acid (5-ASA) is conventionally used as a first line drug for inflammatory bowel disease (IBD). Because 5-ASA is well absorbed in the small intestine, very high dose of 5-ASA is required to deliver it to the large intestine which is a target site. Interestingly, 5-ASA is reported to be transported into the large intestine as well as the small intestine via unknown transport system. In a heterologous expression system using Xenopus oocytes, sodium-coupled monocarboxylate transporter 1 (SMCT1) has been reported to accept 5-ASA as a substrate. Although SMCT1 is found to be expressed in the large intestine, it is unknown whether SMCT1 is responsible for 5-ASA absorption from the large intestine or not. Here we determined the transport characteristics of 5-ASA in the isolated everted sac prepared from mouse large intestine. Na+-dependent uptake of [3H]nicotinate, a substrate for SMCT1, in mouse colon was competitively inhibited by 5-ASA with IC50 value of 2.8 mM. In addition to nicotinate, 5-ASA uptake in mouse colonic mucosa was Na+-dependent and saturable with Michaelis constant (Km) of 2.4 mM. Na+-activation kinetics revealed that the Na+-to-5-ASA stoichiometry was 2:1 and concentration of Na+ necessary for half-maximal transport (K0.5Na) was 36.1 mM. Na+-dependent 5-ASA uptake was competitively inhibited by nicotinate with an inhibitory constant (Ki) of 2.1 mM was comparable to the Km value of Na+-dependent nicotinate uptake (0.99 mM). Furthermore, ibuprofen, a selective SMCT1 inhibitor, was found to have a significantly inhibitory effect on the Na+-dependent 5-ASA uptake in mouse colon (IC50 = 0.19 mM). Taken collectively, these results indicated that SMCT1 in the mouse colonic mucosa is responsible for Na+-dependent 5-ASA uptake.

Combining Chalcones with Donepezil to Inhibit Both Cholinesterases and Aß Fibril Assembly.

The fact that the number of people with Alzheimer's disease is increasing, combined with the limited availability of drugs for its treatment, emphasize the need for the development of novel effective therapeutics for treating this brain disorder. Herein, we focus on generating 12 chalcone-donepezil hybrids, with the goal of simultaneously targeting amyloid-ß (Aß) peptides as well as cholinesterases (i.e., acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)). We present the design, synthesis, and biochemical evaluation of these two series of novel 1,3-chalcone-donepezil (15a-15f) or 1,4-chalcone-donepezil (16a-16f) hybrids. We evaluate the relationship between their structures and their ability to inhibit AChE/BChE activity as well as their ability to bind Aß peptides. We show that several of these novel chalcone-donepezil hybrids can successfully inhibit AChE/BChE as well as the assembly of N-biotinylated Aß(1-42) oligomers. We also demonstrate that the Aß binding site of these hybrids differs from that of Pittsburgh Compound B (PIB).

Thrombin induces Ca2+-dependent glutamate release from RPE cells mediated by PLC/PKC and reverse Na+/Ca2+ exchange.

Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.

Widespread Changes in Positive Allosteric Modulation of the Muscarinic M1 Receptor in Some Participants With Schizophrenia.

BACKGROUND: Preclinical and some human data suggest allosteric modulation of the muscarinic M1 receptor (CHRM1) is a promising approach for the treatment of schizophrenia. However, it is suggested there is a subgroup of participants with schizophrenia who have profound loss of cortical CHRM1 (MRDS). This raises the possibility that some participants with schizophrenia may not respond optimally to CHRM1 allosteric modulation. Here we describe a novel methodology to measure positive allosteric modulation of CHRM1 in human CNS and the measurement of that response in the cortex, hippocampus, and striatum from participants with MRDS, non-MRDS and controls. METHODS: The cortex (Brodmann's area 6), hippocampus, and striatum from 40 participants with schizophrenia (20 MRDS and 20 non-MRDS) and 20 controls were used to measure benzyl quinolone carboxylic acid-mediated shift in acetylcholine displacement of [3H]N-methylscopolamine using a novel in situ radioligand binding with autoradiography methodology. RESULTS: Compared with controls, participants with schizophrenia had lower levels of specific [3H]N-methylscopolamine binding in all CNS regions, whilst benzyl quinolone carboxylic acid-modulated binding was less in the striatum, Brodmann's area 6, dentate gyrus, and subiculum. When divided by subgroup, only in MRDS was there lower specific [3H]N-methylscopolamine binding and less benzyl quinolone carboxylic acid-modulated binding in all cortical and subcortical regions studied. CONCLUSIONS: In a subgroup of participants with schizophrenia, there is a widespread decreased responsiveness to a positive allosteric modulator at the CHRM1. This finding may have ramifications it positive allosteric modulators of the CHRM1 are used in clinical trials to treat schizophrenia as some participants may not have an optimal response.

Regulation of the Serotonergic System by Kainate in the Avian Retina.

Serotonin (5-HT) has been recognized as a neurotransmitter in the vertebrate retina, restricted mainly to amacrine and bipolar cells. It is involved with synaptic processing and possibly as a mitogenic factor. We confirm that chick retina amacrine and bipolar cells are, respectively, heavily and faintly immunolabeled for 5-HT. Amacrine serotonergic cells also co-express tyrosine hydroxylase (TH), a marker of dopaminergic cells in the retina. Previous reports demonstrated that serotonin transport can be modulated by neurotransmitter receptor activation. As 5-HT is diffusely released as a neuromodulator and co-localized with other transmitters, we evaluated if 5-HT uptake or release is modulated by several mediators in the avian retina. The role of different glutamate receptors on serotonin transport and release in vitro and in vivo was also studied. We show that L-glutamate induces an inhibitory effect on [3H]5-HT uptake and this effect was specific to kainate receptor activation. Kainate-induced decrease in [3H]5-HT uptake was blocked by CNQX, an AMPA/kainate receptor antagonist, but not by MK-801, a NMDA receptor antagonist. [3H]5-HT uptake was not observed in the presence of AMPA, thus suggesting that the decrease in serotonin uptake is mediated by kainate. 5-HT (10-50 µM) had no intrinsic activity in raising intracellular Ca2+, but addition of 10 µM 5-HT decreased Ca2+ shifts induced by KCl in retinal neurons. Moreover, kainate decreased the number of bipolar and amacrine cells labeled to serotonin in chick retina. In conclusion, our data suggest a highly selective effect of kainate receptors in the regulation of serotonin functions in the retinal cells.

Roux-en-Y gastric bypass in rat reduces mu-opioid receptor levels in brain regions associated with stress and energy regulation.

Roux-en-Y gastric bypass surgery (RYGB) is the most common and effective weight loss procedure for severe obesity. However, a significant increase in addictive behaviors and new-onset substance use disorder (SUD) are sometimes observed post-surgery. The endogenous opioid system is known to play a major role in motivated behavior and reward, as well as the abuse of substances, including alcohol, tobacco, opioids and highly palatable foods. Here, we examined the effects of RYGB on mu-opioid receptor levels in the brain. Male Sprague-Dawley rats were assigned to one of four groups: standard diet with sham surgery (control), ad libitum high-energy high-fat (HF) diet with sham surgery, calorie restricted HF diet with sham surgery (Sham-FR), or HF diet with RYGB surgery. Control and HF groups were fed their respective diets for 8 weeks, with surgery performed on the eighth week. After 9 weeks on their respective diets post-surgery, animals were sacrificed for mu-opioid receptor autoradiography using the [3H] [D-Ala2,N-Me-Phe4-Gly5-ol]- enkephalin (DAMGO) ligand. Rats with RYGB showed reduced DAMGO binding in the central amygdala compared to sham-operated HF diet controls, and in the hypothalamus compared to high-fat fed Sham-FR. Diet alone did not change [3H] DAMGO binding in any region. These findings show that RYGB surgery, independent of diet or caloric restriction, decreases mu opioid signaling in specific regions important for stress and energy regulation. Thus, RYGB surgery may lead to greater stress sensitivity via downregulated mu opioid signaling in the central amygdala, which may contribute to the observed increased risk in some subjects for addictive behavior.

Histamine H3 receptor activation reduces the impairment in prepulse inhibition (PPI) of the acoustic startle response and Akt phosphorylation induced by MK-801 (dizocilpine), antagonist at N-Methyl-d-Aspartate (NMDA) receptors.

We have investigated the effect of the local activation of histamine H3 receptors (H3Rs) in the rat prefrontal cortex (PFCx) on the impairment of pre-pulse inhibition (PPI) of the startle response induced by the systemic administration of MK-801, antagonist at glutamate N-Methyl-d-Aspartate (NMDA) receptors, and the possible functional interaction between H3Rs and MK-801 on PFCx dopaminergic transmission. Infusion of the H3R agonist RAMH (19.8 ng/1 µl) into the PFCx reduced or prevented the inhibition by MK-801 (0.15 mg/kg, ip) of PPI evoked by different auditory stimulus intensities (5, 10 and 15 dB), and the RAMH effect was blocked by the H3R antagonist/inverse agonist ciproxifan (30.6 ng/1 µl). MK-801 inhibited [3H]-dopamine uptake (-45.4 ±â€¯2.1%) and release (-32.8 ±â€¯2.6%) in PFCx synaptosomes or slices, respectively, and molecular modeling indicated that MK-801 binds to and blocks the rat and human dopamine transporters. However, H3R activation had no effect on the inhibitory action of MK-801 on dopamine uptake and release. In PFCx slices, MK-801 and the activation of H3Rs or dopamine D1 receptors (D1Rs) stimulated ERK-1/2 and Akt phosphorylation. The co-activation of D1Rs and H3Rs prevented ERK-1/2 and Akt phosphorylation, and H3R activation or D1R blockade prevented the effect of MK-801. In ex vivo experiments, the intracortical infusion of the D1R agonist SKF-81297 (37 ng/1 µl) or the H3R agonist RAMH increased Akt phosphorylation, prevented by D1R/H3R co-activation. These results indicate that MK-801 enhances dopaminergic transmission in the PFCx, and that H3R activation counteracts the post-synaptic actions of dopamine.

Presynaptic control of [3H]-glutamate release by dopamine receptor subtypes in the rat substantia nigra. Central role of D1 and D3 receptors.

Substantia nigra pars reticulata is the output station in basal ganglia; its GABAergic neurons control the activity of thalamo-cortical premotor nuclei, thus controlling motor behavior. D1-like and D2-like presynaptic dopamine receptors on subthalamo-nigral afferents by modulation of glutamate release change the firing rate of nigral neurons; however, their relative contribution to the control of glutamate release and their pharmacological properties have not been studied. This is important since the prevalence of the inhibition or stimulation of release determines the firing rate of nigral neurons, therefore motor activity. Here we used depolarization induced [3H]-glutamate release in slices of rat substantia nigra from reserpinized and non-reserpinized rats to explore the relative contribution of the D1-like and D2-like receptor subtypes to the control of glutamate release. We found a significant control of release by D1-like and D3R, and a modest effect of D2R. D4R exerted no effect. Dopamine showed more potency for D3R than for D1-like receptors; however, these latter enhanced release to a greater degree, as shown by the Emax. We also co-activated these to test their interaction; an antagonist interaction of D1-like with D2 and D3R, and an additive between D2 and D3R were found. Pharmacological receptor antagonist effects in release from reserpinized vs. non-reserpinized slices were similar, suggesting that endogenous dopamine stimulates receptors in the same way. These findings suggest differences in the control of glutamate release by different dopamine receptors in the substantia nigra, which could contribute to explaining the effect of dopamine and its agonists on motor behavior.

Cortical and striatal serotonin transporter binding in a genetic rat model of depression and in response to electroconvulsive stimuli.

Depression is a debilitating mental illness and two thirds of patients respond insufficiently to conventional antidepressants. Electroconvulsive therapy (ECT) remains the most effective treatment to alleviate drug-refractory depression, however the neurobiological mechanisms are mostly unknown. The serotonergic system plays an important role in depression and alterations in the serotonin transporter (SERT) are seen both in depression and response to antidepressant pharmacotherapies. The first aim of this study was to investigate SERT density in a genetic rat model of depression, Flinders Sensitive Line (FSL), compared to control Flinders Resistant Line (FRL) and Sprague-Dawley (SD) rats. The second aim was to investigate SERT density in response to electroconvulsive stimuli (ECS), an animal model of ECT. Female rats of each strain were treated with ECS or sham (ear-clip placement with no current) for 10 days before brains were removed, frozen and cut into 20 µm thick sections. SERT density was measured in striatal and cortical regions by quantitative in vitro autoradiography using the SERT-radioligand, [3H]-DASB. Higher SERT density was observed in FSL rats compared to SD rats by 36-48% in motor cortex and striatum under sham conditions. In response to ECS, SD rats displayed a significant effect of treatment, whereas no changes were observed in FRL and FSL rats. Increased SERT binding in FSL rats compared to SD supports a dysfunction of the serotonergic system in depression. The increased SERT density after ECS, seen in SD rats but not FSL rats, suggests a different mechanism of action between depressive-like rats and controls.

Assessing tritium internalisation in zebrafish early life stages: Importance of rapid isotopic exchange.

Tritium (3H) is mainly released into the environment in the form of tritiated water (HTO) by nuclear power plants and nuclear fuel reprocessing plants. To better understand how organisms may be affected by contamination to 3H it is essential to link observed effects to a correct estimation of absorbed dose rates. Due to quick isotopic exchanges between 3H and hydrogen, 3H measurement is difficult in small organisms such as zebrafish embryo, a model in ecotoxicological assay. This work aimed to optimise tritium measurement protocol to better characterise internalisation by early life stages of zebrafish. Zebrafish eggs were exposed at one HTO activity concentration of 1.22 × 105 Bq/mL. This activity was calculated to correspond to theoretical dose rates of 0.4 mGy/h, where some deleterious effects are expected on young fish. A protocol for the preparation of biological samples was adapted from the method classically used to segregate the different forms of tritium in organisms. To deal with very quick isotopic exchanges of 3H with hydrogen, the impacts of washing by non-tritiated water as well as the bias induced by absorbed tritium around organisms on the measured activity concentration were studied. We managed to develop protocols to perform total tritium and total organically bound tritium (OBT) activity concentrations measurements in zebrafish eggs and larvae. The measurement of these both forms allowed the calculation of tissue-free-water-tritium (TFWT). To better understand total tritium internalisation, a study of total tritium kinetics from 4 hpf (hour post-fertilization) to 168 hpf was performed. OBT and TFWT were also assessed to complete the total tritium internalisation kinetics. The internalisation is a rapid phenomenon reaching a steady-state within 24 h after the beginning of contamination for total tritium and TFWT, with concentration factors and TFWT/HTO close to unity. OBT formation seemed to be slower. It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf). To verify absorbed dose rates at key developmental stages (24 hpf eggs and 96 hpf larvae), they were calculated from total tritium activity concentrations after exposure at 1.22 × 105 and 1.22 × 106 Bq/mL, as these two activity concentrations were used to assess effects of tritium in another part of the study. Dose rates calculated from total tritium activity concentrations measured in 24 hpf eggs and 96 hpf larvae were consistent with the nominal ones, which validates the robustness of the protocol developed in the present study.